Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants but the cause is unknown and there are no effective treatments. Using the rat model of NEC, we reported that epidermal growth factor (EGF) markedly reduced the incidence and severity of NEC. The central goal of this proposal is to clarify the molecular mechanism(s) of EGF-mediated reduction of NEC. Accelerated intestinal apoptosis has been shown in patients with NEC, and EGF can protect cells from apoptosis. Our preliminary studies indicate that EGF treatment of NEC regulates pro-survival signaling molecules in the site of NEC injury. Specific Aim 1 will clarify which pro-survival pathways are regulated by EGF treatment of NEC by evaluating EGF treatment (a) on pro-survival and pro-apoptotic genes and proteins in the ileum of neonatal rats with NEC, (b) in waved-2 mice (aberrant EGF-R signaling under non-pathogenic circumstances), (c) in intestinal cell lines where crucial pro-survival and pro-apoptotic genes are over-expressed or blocked and (d) in neonatal rats given EGF-R signaling inhibitors. Recently, we have shown a crucial role of enterohepatic circulation in NEC pathogenesis. Our preliminary data indicate that administration of EGF to neonatal rats with NEC down-regulates luminal bile acid (BA) levels and regulates BA transporters, suggesting an important role of EGF in BA homeostasis. In Specific Aim 2, we will explore the mechanism by which EGF regulates BA transport in the intestine during NEC by evaluating EGF-mediated changes in (a) BA transport in neonatal rats with NEC, (b) IEC lines where essential components of BA transport are over-expressed or blocked, and (c) in waved-2 mice. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the family of EGF-related peptides. Our preliminary results show that HB-EGF reduces the incidence of experimental NEC and enhances EGF-mediated reduction of NEC. Thus, HB-EGF with EGF may provide better protection against NEC through unique nonantagonistic mechanisms. Specific Aim 3 will examine the mechanisms of HB-EGF-mediated reduction of NEC by (a) identifying the optimal route and dose to reduce NEC in neonatal rats, (b) determining the optimal combination of EGF and HB-EGF to reduce NEC, and (c) utilizing an in vitro intestinal injury model to evaluate mechanisms of HB-EGF-mediated reduction of NEC. These studies will elucidate the mechanisms of EGF-mediated reduction of NEC, information that is essential to initiate use of EGF as a preventative or treatment modality for human NEC.